(1) Field of the Invention
The present invention generally relates to regulation of transcription factors. More specifically, the invention relates to a novel SNF2/SWI2 protein family member, SRCAP, that is capable of activating transcription directly as well as interacting with CREB binding protein (CBP) to enhance the ability of CBP to activate transcription.
(2) Description of the Related Art
Transcription factors are well known as proteins that bind to regulatory regions of genes and other proteins to modulate transcription of the genes. Examples of transcription factors relevant to this invention include CREB, c-jun, c-myb, MyoD, E2F1, YY1, TBP, TFIIB, and RNAP II. The action of these transcription factors is affected by co-activators, notably CREB binding protein (CBP).
CBP is a histone acetyltransferase capable of acetylating not only histones but also several transcription factors such as GATA-1 and p53 (Boyes et al., 1998, Nature 396, 594-598; Hung et al., 1999, Cell Biol. 19, 3496-3505; Webster et al., 1999, Mol. Cell. Biol. 19, 3485-3495). CBP also binds to several proteins that also finction as histone acetyltransferases (P/CAF, p/CIP and the p160 co-activators such as SRC-1).
Precisely how CBP interacts with these co-activators and other cellular factors to activate transcription has not been completely elucidated. The notion that CBP interacts with a specific subset of factors at different promoters was first suggested by the work of Korzus et al., 1998, Science 279, 703-707. These authors showed that CBP in conjunction with P/CIP, SRC-1 and P/CAF was required for activation of transcription of a RARE reporter gene by the retinoic acid receptor, whereas only CBP, P/CAF and p/CIP were needed for activation of a CRE reporter gene by CREB and only CBP and P/CAF are required for transcription of a GAS-reporter gene by STAT-1. In addition the HAT activity of each of these co-activators was not needed at each promoter. For example, with the RARE- reporter gene, despite the fact that the pCIP-P/CAF-SRC-1-CBP complex was needed for activation of transcription, only the HAT activity of P/CAF was needed, whereas transcription of the CRE-reporter by the CBP-P/CAF-pCIP complex required the HAT activity of CBP not p/CAF. Thus, the specific transcription factors which CBP binds determine not only the requirement for specific co-activators but whether their HAT activity is also needed. The requirement for a specific HAT function may also be altered depending on what signaling pathways activate transcription. Xu et al. (1998, Nature 395, 301-306) have reported that forskolin activation of Pitl mediated transcription requires the HAT finction of CBP whereas insulin activation of Pitl mediated transcription does not.
Why different HAT activities are needed is unclear but CBP, P/CAF and p/CIP have been shown to have different substrate specificities (Perissi et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96, 3652-3657) suggesting they acetylate a different subset of proteins. The possibility that substrate specificity of these proteins may be regulated is illustrated by the work of Perissi et al. (Id.) who reported that binding of p/CIP to CBP changes the substrate specificity of CBP. Repression of the HAT activity of CBP by the adenoviral 12S E1A protein has been reported by several laboratories and occurs through E1A contacts with the HAT domain of CBP (Charkravati et al., 1999, Cell 96, 393-403, reviewed in Goldman et al., 1997, Recent Prob. Horm. Res. 52, 103-120). In contrast to these findings, recent work by Ait-Si Ali et al. (1998, Nature 396, 184-186) indicates that in some circumstances E1A can stimulate the HAT activity of CBP.
The activity of several kinases has been shown to regulate CBP function. The NGF stimulated kinase, p42/44MAPK, activates CBP mediated transcription by a phosphorylation event which is blocked by the MAPK inhibitor PD 98059. p42/44 MAPK directly associates with CBP and can phosphorylate CBP in vitro (Liu et al., 1999, Neuroreport 10, 1239-1243). Studies have also demonstrated the cyclin E-cyclin dependent kinase 2 (cdk2) complex binds CBP and mediates hyperphosphorylation of CBP at the G1/S boundary (Ait-Si Ali et al., supra). Inhibition of cyclin E-Cdk2 by the cyclin dependent kinase inhibitor p21, results in the activation of NF-kB mediated transcription (Perlins et al., 1997, Science 275, 523-527). The binding of the kinase pp90RSK to CBP has different effects depending on the signaling pathway. pp90RSK-CBP interaction is needed for insulin stimulated transcription, and blocks the ability of CBP to function as a co-activator for cAMP-mediated transcription. The activity of pp90RSK can be mimicked by a kinase defective mutant, indicating association of pp90RSK with CBP is enough to alter its finction (Nakajima et al., 1996, Cell 86,465-474). Other studies have also demonstrated that protein kinase A and CAM kinase II and IV positively modulate the ability of specific domains within CBP to activate transcription. However, the precise mechanism by which this occurs has not been delineated (Swope et al., 1996, J. Biol. Chem 271, 28138-28145, Liu et al., 1998, J. Biol. Chem. 273, 25541-25544).
CBP interacts with several general transcription factors. Swope et al. (Id.) demonstrated that TBP binds in vitro to the N-terminal end of CBP and Abrahams et al. (1993, Oncogene 8, 1639-1647) and Dallis et al. (1997, J. Virol. 71, 1726-1731) have shown that TBP binds CBP in vivo. TFIIB binds to the C-terminal end of CBP and this binding is blocked by the adenoviral protein E1A (Felzien et al., 1999, Mol. Cell. Biol. 19,4261-4266). Deletion of the TBP binding domain from CBP prevents it from acting as a co-activator for CREB whereas deletion of the TFIIB binding site has no effect (Swope et al., Id.). CBP also binds RNA helicase A and this association appears to be required for the interaction of CBP with RNA polymerase II Nakajima et al., 1997, Cell 90, 1107-1112).
Phosphorylation of CREB in the KID domain enhances its ability to activate transcription and is mediated by a number of kinases (CAM kinase H and IV and protein kinase A) in response to biological stimuli such as neuronal signals or increased levels of intracellular cAMP (Gonzalez et al., 1991, Mol. Cell. Biol. 11, 1306-1312; Fiol et al., 1994, J. Biol. Chem. 269, 32187-32193; Sun et al., 1994, Genes Develop 8, 2527-2539). Specific iphosphorylation of serine 133 within CREB promotes association with CBP but studies by Sun and Maurer (1995, J. Biol. Chem. 270, 7041-7044) indicate this phosphorylation is not sufficient for activation of transcription. Other studies have found that phosphorylation of CREB on serines 129 and 142 modulate the ability of CREB to activate transcription (Fiol et al., supra; Sun and Maurer, supra) suggesting they change the way in which CREB and CBP interact perhaps alters the function of CBP (e.g. the HAT activity or binding of other proteins). The ability of CREB to activate transcription is also blocked by the MAPK inhibitor PD 98059 (Seternes et al., 1999, Mol. Endocrinol. 13, 1071-1083). Although the mechanism by which this occurs has not been completely elucidated other studies indicate that PD 98059 completely inhibits the ability of CBP to activate transcription (Liu et al., 1998, J. Biol. Chem. 273, 25541-25544).
Structure-finction studies by Swope et al. (1996, J. Biol. Chem 271, 28138-28145) indicated that deletion of amino acids 272-460 from the amino terminal end of CBP prevent it from acting as a co-activator for CREB. A CBP227-460 peptide containing this deleted region also functions as a strong transcriptional activator when expressed as a Gal chimera (Gal-CBP227-460). In addition, overexpression of a CBP1-460 peptide squelches the ability of full length CBP to function as a CREB co-activator. Collectively, these studies suggest that a factor binding to the amino terminal end of CBP is essential for it to function as a co-activator for CREB. These studies also indicate that CBP contains several regions which are involved in the activation of transcription and which function as transcriptional activators when expressed as gal-CBP chimeras (Id.). Various binding studies have identified a number of co-activators, general transcription factors, kinases, and transcription factors that contact these regions of CBP. A partial list is shown in FIG. 1, where identification of the CBP domain that interacts with SRCAP is newly disclosed herein. See also Goldman et al., 1997, Recent Progress in Hornone Research 52, 103-1221.
A growing body of literature now illustrates the importance of CBP. The most compelling evidence for the importance of CBP has come from naturally occurring and artificially developed mutations. CBP appears to be critical in the development of the CNS, since a mutation in CBP has been associated with mental retardation in humans (Rubinstein-Taybi syndrome) (Petrij et al., 1995, Nature 376, 348-351). This hypothesis is strengthened by the finding that transgenic mice bearing similar CBP mutations have clinical features associated with Rubinstein-Taybi syndrome including: deficiency in long term memory, growth retardation, cardiac anomalies and skeletal abnormalities, and defects in hematopoiesis and vasculo-angiogenesis (Oike et al., 1999, Blood 93, 2771-2779; Oike et al., 1999, Genetics 8, 387-396). Mutations of CBP have also been found in a subset of patients with acute myeloid leukemia that have a t(8; 16)(p 11: p 13) chromosomal translocation which fuses CBP to the acetyltransferase MOZ (Burrow et al., 1996, Nature Genetics 383, 99-103).
The observation that mutations in CBP cause many distinct physiological defects is not surprising, since CBP serves as a co-activator for a wide variety of transcription factors. For example, CBP interacts with estrogen, progesterone, retinoic acid, vitamin D, glucocorticoid, and PPARxcex3 receptors (reviewed in Mckenna et al., 1999, Endocrine Reviews 20, 321-344), which are important for sexual maturation, bone development, lipid metabolism and regulation of energy metabolism. CBP interacts with transcription factors that direct pancreatic islet morphogenesis (NeuroDI/BETA2, Sharma et al., 1999, Mol. Cell. Biol. 19, 704-713) and liver development (HNF4, Dell et al., 1999, J. Biol. Chem. 274, 9013-9021). CBP also serves as a co-activator for a number of transcription factors activated by growth factors such CREB, jun, fos, smad proteins and NF-kappaB, as well as a number of constitutively active transcription factors such as c-myb, NF-1 C, GATA-1 and p53 (Dai et al., 1996, Genes and Develop. 10, 524540; Chaudry et al., 1999, J. Biol. Chem. 274, 7072-7081; Hung et al., 1999, Cell Biol 19, 3496-3505; Lambert et al., 1998, J. Biol. Chem. 273, 33048-33053).
Competition or xe2x80x9ccross talkxe2x80x9d between transcription factors for the limiting amount of CBP present in the cell has been reported to be a control mechanism for cellular response to different signaling pathways. For example, competition between NF-kappaB and the tumor suppressor p53 for CBP determines whether p53 mediated apoptosis occurs (Webster et al., 1999, Mol. Cell. Biol. 19, 3485-3495). Competition between GR and NF-kappaB has also been reported as a control mechanism for glucocorticoid mediated repression of NF-kappaB-mediated inflammation (Sheppard et al., 1998, J. Biol. Chem. 273, 29291-29294). In LNcAP cells, overexpression of the androgen receptor can block AP-1 mediated transcription (Fronsdal et al., 1999, J. Biol. Chem. 273, 31583-31859).
CBP is critical for the functioning of several viruses that impact human health. This includes human T cell lymphotrophic virus (HTLV-1), which recruits CBP to viral promoters through interaction of the viral protein TAX with CREB (Kwok et at., 1996). CBP also interacts directly with the viral transactivator protein Tat of human immunodeficiency virus type I (mHV-1) (Hottiger and Nabel, 1998, J. Virol. 72, 8252-8256). In humans, adenoviruses cause several diseases including: acute follicular conjunctivitis, pharyngoconjunctivial fever, epidermic keratoconjunctivitis, acute hemorrhagic cystitis, cervicitis, infantile diarrhea, and respiratory tract infections in children. Immune compromised individuals such as those who have undergone bone or organ transplant or who have AIDS are particularly susceptible to adenoviral caused diseases (reviewed in Chapter 65 of Zinsser, 1992, Microbiology, 20th Ed., Joklik et al., Eds., Appleton and Lange). CBP interacts with several isoforms of the adenoviral protein EIA to mediate repression or activation of transcription (Felzien et al., 1999, Mol. Cell. Biol. 19,42616). CBP also functions as a co-activator for the zta protein of Epstein-Barr virus (Zerby et al., 1999, Moll. Cell. Biol. 19, 1617-1626). Epstein-Barr virus infections have been associated with fatal lymphoproliferation in immune deficient patients and the development of Burkitt""s lymphoma (reviewed in Zinsser Microbiology, 1992, supra). CBP has also been indirectly implicated in the transcriptional regulation of other viruses through interaction with CREB. For example, CREB binds to the enhancer of the IE1/2 gene of human cytomegalovirus (Lang et al., 1992, Nucl. Acids Res. 20, 3287-3295), an important pathogen in immunosuppressed patients such as transplant recipients and AIDS patients (Drew, 1988, J. Infect. Dis. 158, 449456). CREB also binds the hepatitis B virus (HBV) enhancer element when complexed with the HBV protein, pX (Maguire et al., 1991, Science 252, 842-844). A possible role of CBP in some forms of cancer is suggested by the observation that CBP is part of a multi-subunit complex with the breast cancer tumor suppressor BRCA1 and RNA polymerase II (Neish et al., 1998, Nucl. Acids Res. 26, 847-853). In some forms of acute myeloid leukemia a t(7;11)(p15: p15) translocation results in the NUP98-HOXA9 fusion protein, which is a strong transcriptional activator that uses CBP as a co-activator (Kasper et al., 1999).
CREB and, therefore, CBP have been implicated as possible effectors of a number of physiological responses in the central nervous system. In rats, acute morphine treatment increases the level of activated CREB in the locus coeruleus. This effect is attenuated with chronic treatment. Acute morphine withdrawal increases the level of activated CREB. Thus, regulation of genes by CREB may contribute to changes leading to addiction (Guitart et al, 1992, J. Neurochem. 58, 1168-1171). Furthermore, the level of phosphorylated CREB increases in the suprachiamatic nuclei in response to light, suggesting it may be important for the entrainment of the pacemaker that controls hormonal and behavioral cycles (Ginty et al., 1993, Science 260, 238-241). In aplysia, the regimented application of serotonin to sensory neurons can be used to induce long-term memory. Analogs of cAMP are able to elicit some of the same responses suggesting CREB is involved. This hypothesis is supported by the finding that serotonin induces the phosphorylation of CREB (Kaang et al., 1993, Neuron 10, 427-435) and that microinjection of the oligonucleotides containing the binding site for CREB blocks serotonin induced long term memory (Dash et al., 1990, Nature 345, 718-721). Mice containing deletions in certain forms of CREB also appear to have defects in learning (Bourtchuladze et al., 1994, Cell 79, 59-68). Several lines of experiments also indicate CREB is involved in mediating the long-term effects of activity dependent plasticity at glutamatergic neurons (reviewed in Ghosh and Greenberg, 1995, Science 268, 239-247). A role of CBP is suggested by the recent studies by Hu et al. (1999, Neuron 22, 1-22), which indicate activation of glutamatergic neurons leads to an increase in the ability of CBP to activate transcription. This appears to be mediated by CaM kinases II and IV activated in response to increased calcium levels following depolarization and leads to phosphorylation of CBP (Hu et al., Id.).
Based on the importance of CBP in activating transcription, it would be desirable to identify other proteins that serve as transcriptional co-activators with CBP.
Accordingly, the inventors have succeeded in discovering a protein, called SRCAP, for Snf2 Related CBP Activator Protein, that is a transcriptional co-activator for CREB binding protein (CBP). SRCAP is unlike all other known CBP co-activators in that it also is a Snf2 family member, and, as such, has ATPase activity. SRCAP also is capable of activating transcription without CBP and is also capable of interacting with DEAD box helicases, adenoviral DBP protein, xcex2-actin and nuclear receptors. SRCAP is also capable of DNA binding. Among the compositions also provided herein are fragments of SRCAP, polynucleotides encoding SRCAP or SRCAP fragments, complements of the SRCAP polynucleotides or fragments, and antibodies that specifically bind to SRCAP. These compositions are useful for, e.g., enhancing or suppressing CBP mediated transcriptional co-activation, and treatment of diseases involving inappropriate transcriptional activation that can be mediated by SRCAP or by CBP.
Thus, some embodiments of the present invention is directed to an isolated and purified SRCAP polypeptide. The polypeptide comprises the amino acid sequence of SEQ ID NO:2, or a conservatively substituted variant thereof, and wherein the polypeptide has ATPase activity and is capable of activating transcription. An example of a SRCAP polypeptide is provided as SEQ ID NO:2.
The present invention is also directed to an isolated and purified naturally occurring polypeptide comprising an amino acid sequence that has at least about 80% sequence homology to SEQ ID NO:2, wherein the polypeptide has ATPase activity and is capable of activating transcription. In preferred embodiments, the amino acid sequence that has at least about 90% sequence homology to SEQ ID NO:2.
In additional embodiments, the invention is directed to a polypeptide comprising at least 15 contiguous amino acids of any of the SRCAP polypeptides described above. In certain aspects, the polypeptide has at least one of the following SRCAP activities: ATPase, CREB binding protein (CBP) interaction and transcriptional co-activation, transcriptional activation without CBP, and DNA binding.
The present invention is also directed to a SRCAP chimera having a part of one of the above SRCAP polypeptides covalently attached to an amino acid sequence from a naturally occurring protein that is not a SRCAP. In preferred embodiments, the protein that is not SRCAP is a GAL4.
The present invention is further directed to a polynucleotide comprising a nucleic acid encoding any of the above-described SRCAP polypeptides. An example is SEQ ID NO:11. Complementary sequences to any of these polynucleotides are also provided, as are sequences that are capable of selectively hybridizing to the above sequences, as well as fragments of any of the polynucleotide sequences, where the fragment is at least 45 nucleotides long. These polynucleotides can be part of a vector used, e.g., to replicate the polynucleotide or express the encoded polypeptide. The vectors can also be used to create recombinant cells comprising any of the invention polynucleotides, and/or encoding any of the invention polypeptides.
The cells can be, e.g., an E. coli cell or any other appropriate cell, including a mammalian cell. The cell can also be part of a living animal, including a mammal.
The above polynucleotides can also be capable of hybridizing to a SRCAP mRNA and, as such, can serve as an antisense agent. Ribozymes capable of specifically cleaving any of the invention polynucleotides is also provided, as are polynucleotides encoding any of the previously described chimeras.
In additional embodiments, the present invention is also directed to antibodies that specifically bind to any of the above described polypeptides. In a preferred embodiment, the antibody binds to amino acids 2733-2971 of SEQ ID NO:11.
Additionally, the present invention is directed to a method of activating transcription in a cell. The method comprises treating the cell with any appropriate polypeptide, polynucleotide, or antibody of the invention. In preferred embodiments, the cell is in a mammal, which includes humans. In some aspects, the method further comprises implanting the cell into a mammal after treating the cell.
The present invention is also directed to methods of detecting a SRCAP in a sample. The methods comprise treating the sample with one of the above-described antibodies, then determining whether the antibody specifically binds to a component of the sample. In these methods, specific binding indicates the presence of SRCAP in the sample. The sample can be from a mammal, including humans. In preferred embodiments, the determining step is by ELISA, RIA, or methods that include an electrophoresis step, such as western blot. For these methods, the sample can also be an intact tissue such as a tissue section.
In additional embodiments, the present invention is directed to a method of detecting any of the invention polynucleotides in a sample. The method comprises treating the sample with a second polynucleotide that specifically hybridizes with the polynucleotide of interest, then determining if the second polynucleotide specifically hybridized with a component of the sample. In some embodiments, the treating step is by a Southern hybridization method or a northern hybridization method.
The present invention is also directed to an additional method of detecting any of the invention polynucleotides in a sample. The method comprises treating the sample with a second polynucleotide that specifically hybridizes with the polynucleotide of interest, then performing a polymerase amplification method such as PCR or RT/PCR.
The present invention is further directed to methods of enhancing CREB binding protein (CBP)-mediated activation of transcription in a cell. The methods comprise treating the cell with one of the polypeptide, polynucleotide, or antibody compositions described above. As with previous methods, the cell can be part of a mammal, including a human.
The present invention is still further directed to a method for identifying a compound that modulates SRCAP protein function. The method comprises determining whether the candidate modulator compound alters the interaction of the SRCAP with CBP.
Additionally, the invention is directed to methods for identifing a compound that modulates SRCAP function. The methods comprise assessing the activity of SRCAP as an ATPase, in CBP interaction and transcriptional co-activation, in transcriptional activation without CBP, or in DNA binding in the presence of the candidate modulator compound.
In additional embodiments, the present invention is directed to a method for treating a patient having a disease involving a function affected by SRCAP protein. The method comprises administering an invention polypeptide or polynucleotide to the patient. In preferred embodiments, the patient is a mammal, including humans. Preferred functions are insufficient transcription of a gene selected from the group consisting of a gene mediated by CBP co-activation, a DEAD box RNA dependent helicase, an adenoviral DBP protein, xcex2-actin and a nuclear receptor.
In further embodiments, the present invention is directed to a method for treating a patient having a disease mediated by SRCAP-activated transcription. The method comprises administering to the patient a compound that decreases SRCAP activity in the patient. Preferred compounds here are antibodies, antisense polynucleotides or ribozymes. In preferred embodiments, the disease is a virus infection, for example infection with adenovirus, HCV, HTLV-1, HIV-1, Epstein-Barr virus, cytomegalovirus or hepatitis B virus.